Composition containing eosinophil cationic protein

ABSTRACT

A therapeutic composition for a disease caused by a failure in the survival, proliferation and/or differentiation of a cell which contains eosinophil cationic protein and other components; and a medium composition for promoting the survival, proliferation and/or differentiation of a cell.

TECHNICAL FIELD

The invention of this application relates to a composition containingeosinophil cationic protein. More particularly, this application relatesto a therapeutic composition and a cell culture medium compositionutilizing the novel physiological activity of eosinophil cationicprotein, and a screening method by using the physiological activity ofeosinophil cationic protein as an indicator.

BACKGROUND ART

Eosinophil cationic protein (hereinafter referred to as “ECP”) is aprotein whose expression is increased in accordance with the activationof an eosinophil, and is present in a basic granule (Proc. Natl. Acad.Sci. USA. 1986, 83 (10):3146-3150). It is known that ECP has activitiesof killing parasites, neurotoxin, inhibition of lymphocyteproliferation, sterilization, histamine release, ribonuclease,shortening the clotting time, and the like (Nippon Rinsho 1993, 51 (3):60 (606)). In particular, since its histamine releasing activity inducesan allergic reaction, an antiallergic drug whose pharmacological actionis to inhibit the ECP expression has been proposed (JP-T-2002-500506(the term “JP-T” as used herein means a published Japanese translationof a PCT patent application)). In addition, it has been proposed that aneosinophil cell line including ECP is utilized for screening of a drugfor asthma or an antiallergic drug (JP-A-05-111382).

As described above, it has been recognized that the physiologicalactivity of ECP is toxicity or proliferation inhibition against bacteriaor animal cells so far.

On the contrary, the inventors of this application found that ECP has anovel physiological activity such as the survival or differentiation ofan animal cell.

An object of the invention of this application is to provide a novelcomposition utilizing the novel activity of ESP that the inventorsfound.

In addition, another object of this application is to provide a novelmethod for developing a remedy whose pharmacological action is topromote the survival or differentiation of a cell by using the novelactivity of ECP as an indicator.

DISCLOSURE OF THE INVENTION

This application provides, as a first invention for achieving theforegoing objects, a composition which is a therapeutic composition fora disease caused by a failure in the survival, proliferation and/ordifferentiation of a cell characterized by containing eosinophilcationic protein and a pharmacological component.

In the composition of this first invention, a preferred embodiment isthat the disease caused by a failure in the survival, proliferationand/or differentiation of a cell is a heart disease, bone disease orneurodegenerative disease.

Further, this application provides, as a second invention, a compositionwhich is a medium composition for promoting the survival, proliferationand/or differentiation of a cell and containing eosinophil cationicprotein and a cell biological component.

Further, this application provides, as a third invention, a screeningmethod, which is a method of screening an active ingredient substance ofa therapeutic composition for a disease caused by a failure in thesurvival, proliferation and/or differentiation of a cell, characterizedby bringing a candidate substance into contact with a cell andspecifying, as a target substance, a substance for promoting thesurvival and/or differentiation of a cell at the same level or higherthan eosinophil cationic protein.

In this third invention, a preferred embodiment is that the cell is anerve cell, bone cell, myocardial cell or fibroblast.

In other words, the inventors of this application found the followingnovel activities of ECP, which was conventionally considered to havephysiological activities of cytotoxicity and inhibition of cellproliferation.

(1) Promotion of fibroblast proliferation and promotion of stress fiberformation

(2) Maturing of muscle fiber of a myocardial cell and increase in thenumber of heartbeats

(3) Improvement of survival rate of nerve cells in a low-serum medium orserum-free medium

(4) Promotion of fibroblast differentiation

In addition, the foregoing activities are inhibited or lost by aninhibitor against a substrate of low molecular weight G-proteinRho-kinase (ROCK) which plays an important role in the signaltransduction pathway in a cell. Therefore, it was found that ECP affectsthe signal transduction pathway in a cell and provides the foregoing (1)to (4) activities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results obtained by studying the effects of ECP on theproliferation of cell lines derived from normal tissues. The closedcircles indicate BALB/c 3T3 cells, the open triangles indicate A10cells, the open squares indicate HC-11 cells, and the open circlesindicate HUVEC cells.

FIG. 2 shows the results obtained by studying the dose-dependent effectsof ECP on the proliferation of BALB/c 3T3 cells.

FIG. 3 shows the results obtained by studying the time-course effects ofECP on the proliferation of BALB/c 3T3 cells. The closed circlesindicate EPC, the closed squares indicate RNase and the open squaresindicate the control.

FIG. 4 shows the results (phase-contrast microscopic images) obtained bystudying the effects of ECP on the proliferation of BALB/c 3T3 cells ina low-serum medium.

FIG. 5 shows the results (confocal laser scanning microscopic images)obtained by studying the effects of ECP on the formation of BALB/c 3T3cytoskeletal molecule in a low-serum medium.

FIG. 6 shows the results (confocal laser scanning microscopic images)obtained by studying the effects of ECP+bFGF on the formation of BALB/c3T3 cytoskeletal molecule in a low-serum medium.

FIG. 7 shows the results (confocal laser scanning microscopic images)obtained by studying the effects of ECP and a ROCK inhibitor on thecytoskeleton formation of BALB/c 3T3 cells.

FIG. 8 shows the results (confocal laser scanning microscopic images)obtained by studying the effects of ECP on the cytoskeleton formation inmyocardial cell derived from a newborn rat.

FIG. 9 shows the results obtained by studying the effects of ECP on thenumber of heartbeats of myocardial cell derived from a newborn rat.

FIG. 10 shows the results obtained by studying the effects of ECP and aROCK inhibitor on the number of heartbeats of myocardial cell.

FIG. 11 shows the results obtained by studying the effects of ECP on thesurvival of nerve like PC12 cells in a serum-free medium. The columns 1,2, 3, and 4 correspond to the mean values in the cases of before ECPaddition, addition of ECP at 0 ng/ml, 10 ng/ml and 1000 ng/ml,respectively.

FIG. 12 shows the results obtained by studying the effects of ECP on thealkali phosphatase activity in MC3T3-E1 cells derived from calvarialbone of a rat.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention of this application is based on the following newfindings. Hereunder, with regard to each invention of this application,embodiments will be explained in detail.

ECP used in each invention of this application can be isolated from acell (leukocyte cell or hematopoietic stem cell) of a variety of mammalssuch as human by a known method. In addition, it can be also produced bychemical synthesis using a known solid-phase peptide synthetic methodbased on the amino acid sequence (human ECP: GenBank/X15161, chimpanzee:GenBank/AF294028, gorilla: GenBank/U24097) or the like. Further, it canbe obtained as a recombinant ECP by expressing a polynucleotide encodingeach peptide in an in vitro transcription/ translation system or anappropriate host-vector system. The polynucleotide (e.g., ECP cDNA) canbe obtained by a method of screening an existing cDNA library with theuse of an oligonucleotide probe produced based on the nucleotidesequence information in the foregoing GenBank database, or a knownmethod such as the RT-PCR method using an oligonucleotide primer.

For example, in the case of producing a recombinant ECP in an in vitrotranscription/translation system, the foregoing polynucleotide isinserted into a vector with an RNA polymerase promoter to produce anexpression vector, and this vector is added to an in vitro translationsystem such as a rabbit reticulocyte lysate or a wheat germ extractcontaining an RNA polymerase corresponding to the promoter. As the RNApolymerase promoter, T7, T3, SP6 and the like can be exemplified. As thevector containing such an RNA polymerase promoter, pKA1, pCDM8, pT3/T718, pT7/3 19, pBluescript II and the like can be exemplified.

In the case of expressing such a recombinant ECP in a microorganism suchas E. coli, a recombinant expression vector is produced by recombiningthe foregoing DNA fragment into an expression vector having an origincapable of replication in a microorganism, a promoter, ribosome bindingsite, DNA cloning site, terminator or the like, and a fusion peptide isisolated from the culture. As the expression vector for E. coli, a pUCsystem, pBluescript II, pET expression system, pGEX expression systemand the like can be exemplified.

In the case of expressing such a recombinant ECP in a eukaryotic cell, arecombinant expression vector is produced by inserting the foregoingfusion polynucleotide into an expression vector for a eukaryotic cellhaving a promoter, splicing site, poly(A) addition site or the like, andthe recombinant vector is introduced into a eukaryotic cell, whereby afusion peptide can be expressed in a transformed eukaryotic cell. As theexpression vector, pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBVvector, pRS, pcDNA3, pMSG, pYES2 and the like can be exemplified. As theeukaryotic cell, a cultured mammalian cell such as a monkey kidney cellCOS7 and Chinese hamster ovary cell CHO, budding yeast, dividing yeast,silkworm cell, African clawed frog egg cell and the like are usuallyused, however, any eukaryotic cell may be used so long as it is able toexpress a desired protein.

For the introduction of the expression vector into a host cell, a knownmethod such as an electroporation method, a calcium phosphate method, aliposome method, and a DEAE dextran method can be employed.

In order to isolate or purify a recombinant ECP from the culture afterexpressing the fusion peptide in a prokaryotic cell or a eukaryoticcell, it can be performed by combining known separation procedures. Forexample, these procedures include treatment with a denaturation reagentsuch as urea or with a surface active agent, ultrasonic treatment,enzymatic digestion, salting-out and solvent precipitation method,dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE,isoelectric focusing electrophoresis, ion exchange chromatography,hydrophobic chromatography, affinity chromatography, reversed phasechromatography and the like.

Incidentally, ECP is a secretory protein, and in the case of, forexample, human ECP, 23-amino acid sequence at the N-terminal side is asecretory signal sequence. Therefore, in the case where a recombinantECP is expressed in a prokaryotic cell or a eukaryotic cell, it ispreferred that an active region at the C-terminal side from thesecretory signal sequence (e.g., C-terminal residues from Arg-28) isexpressed in consideration of the recovery efficiency of the recombinantECP from the culture.

The composition of the first invention is a medical compositioncharacterized by containing the foregoing ECP and a pharmacologicalcomponent, and is used for a cure of a human disease caused by a failurein the survival, proliferation and/or differentiation of a cell. Inother words, such a disease is developed by an aberration in a normallifecycle (proliferation, differentiation, apoptosis or the like) of avariety of tissue cells of the body, and a variety of diseases caused byheredity or various cytotoxic substances can be used as a target. Inparticular, it can be used for a complete cure, remission or improvementof symptom of a heart disease, bone disease or neurodegenerativedisease. Examples of the heart disease include cardiac infarction causedby such as aberration or degeneration of myocardial cytoskeletonformation, myocarditis, cardiomyopathy (such as dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andobliterative cardiomyopathy), myocardial fibrosis and the like. Examplesof the bone disease include particularly osteoporosis caused by afailure in differentiation of a bone-forming cell (osteoblast) andperiodontal disease. In addition, this therapeutic composition can beused for osseous tissue regeneration after fracture. Examples of theneurodegenerative disease include Alzheimer disease caused bydegeneration or apoptosis of a nerve cell, dementia senilis, Downsyndrome, Parkinson's disease, Creutzfeldt-Jakob disease, amyotrophiclateral sclerosis, neuromyopathy and the like.

The “pharmacological components”, which is a component of the medicalcomposition of the first invention means firstly, a variety of carriersto be used in the usual drug production. The carrier can beappropriately selected from a wide range according to the types of atarget disease or a dosage form of a drug, however, it is desired thatthe medical composition of this invention is in a unit dosage form whichcan be administered orally or by injection. Particularly, in the case ofadministration by injection, topical injection, intraperitonealinjection, selective intravenous infusion, intravenous injection,hypodermic injection, organ perfusate infusion or the like can beadopted.

An oral liquid preparation such as a suspension or a syrup can beproduced by using water, a saccharide such as sucrose, sorbitol andfructose, a glycol such as polyethylene glycol, an oil such as sesameoil and soybean oil, an antiseptic such as alkyl p-hydroxybenzoate, aflavor such as strawberry flavor, peppermint, etc.

A powder, pill, capsule, and tablet can be formulated by using a diluentsuch as lactose, glucose, sucrose and mannitol, a disintegrator such asstarch and sodium alginate, a lubricant such as magnesium stearate andtalk, a binder such as polyvinyl alcohol, hydroxypropyl cellulose, andgelatin, a surface active agent such as a fatty acid ester, aplasticizer such as glycerin, etc. A tablet and a capsule are apreferred unit dosage form for the composition of this invention in thatit is easy to administer. In the production of a tablet or a capsule, asolid pharmaceutical carrier is used.

In addition, a solution for injection can be formulated by using acarrier comprising a salt solution, a glucose solution or a mixture of asalt water and a glucose solution, a variety of buffers or the like.Alternatively, it is formulated in a powder form, and an injectionsolution may be prepared by mixing the powder with the foregoing liquidcarrier at the point of use.

The administration amount of the medical composition of this inventionvaries depending on the age or body weight of a patient, symptom,administration route or the like, however, the amount that allows theblood concentration of ECP to be about 10 nmol to 0.1 mmol, preferablyabout 5 nmol to 0.5 mmol may be administered.

A second pharmacological components is a component for allowing ECP tobe in a form that can be introduced into a cell. For example, thispolypeptide is mixed with a solution which does not change the structureor the function of this polypeptide and is pharmacologically acceptable,whereby the composition can be prepared. Such a composition can beintroduced into a target cell by, for example, an intracellularintroduction method by a microinjection method or by an intracellularintroduction method using a lipid (e.g., BioPORTER (Gene TherapySystems, USA), or Chariot (Active Motif, USA), or the like).

A third pharmacological components is a component for allowing apolynucleotide encoding ECP to be in a form that can be introduced intoa cell. In other words, it may be performed by incorporating thepolynucleotide into an expression vector for a eukaryotic cell, andincorporating this vector into, for example, a hollow nanoparticlepresenting a biorecognizable molecule, retrovirus, adenovirus,adeno-associated virus or the like. Such a composition can be introducedinto a target cell in the body by a method of gene therapy.

The second invention of this application is a medium compositioncontaining eosinophil cationic protein and a cell biological component,and can be used for promoting the survival, proliferation and/ordifferentiation of a cultured cell.

The “cell biological component” is a component essential for thesurvival, proliferation, differentiation or the like of a cell andspecifically, it is a component constituting a usual medium for ananimal cell. Specific examples include a buffer (phosphate, sodiumbicarbonate, carbon dioxide gas or the like), a salt, glucose, avitamin, an organic substance such as an amino acid, serum (fetal bovineserum: FBS), a nutritional factor (growth factor in serum: GFS) and thelike. The medium composition of this invention can be produced byappropriately mixing such a component and ECP. The content of eachcomponent can be set at the same level as the usual culture medium foran animal. The content of ECP can be arbitrarily set depending on thetype of a cell or the object, however, it can be set to about 0.001 to10μ per 1 ml of medium.

As the “cell”, a cell which is usually used as a target for an animalcell can be used, however, particularly a nerve cell, bone cell,myocardial cell, fibroblast and the like are preferred. In addition,such a cell may be a primary culture cell isolated from an animaltissue, passage culture cell, established cell line, or transformed cellinto which a foreign gene has been introduced. “Culture” can be carriedout in a floating culture system in the case of a floating cell, and ina monolayer culture system or three-dimensional culture system in thecase of an adhesive cell. In addition, it may be carried out in a hollowfiber culture system in a hollow polymeric tube.

The culture using the medium composition of the second invention allowsa cultured cell to survive over a long period of time, or to bedifferentiated for expressing a function. Therefore, the culture systemusing this composition is useful in elucidating a cell biological eventsuch as the survival, cytoskeleton formation, proliferation ordifferentiation of a cell, or the signal transduction cascade regulatingthese events (especially, Rho kinase pathway). In addition, since thiscomposition allows a cell to be differentiated in a state of expressinga function, or to survive over a long period of time, this compositioncan be utilized in the production (bioreactor or the like) of a usefulsubstance.

Further, the medium composition of this invention allows a nerve cell orthe like to survive over a long period of time even in a low-serum or aserum-free condition by containing ECP. Therefore, because a cellfunction can be analyzed in a condition of low protein or free ofprotein except for ECP, it is useful in a system for screening a proteinfactor, etc. which affects a specific function of a cell.

The third invention of this application is a screening method forspecifying a novel factor other than ECP, which has an action ofpromoting the survival, proliferation and/or differentiation of a cell.In other words, a candidate substance is brought into contact with acell, and the survival time or differentiation state of the cell ismeasured. Then, in the case where the measurement value is equal to orhigher than the measurement value when ECP was brought into contact witha cell, the tested candidate substance can be determined to be a targetfactor. The survival of a cell can be measured by a known method such asthe trypan blue staining method. The proliferation of a cell can bemeasured by counting the number of living cells after being cultured fora predetermined period. The differentiation of a cell can be measured bythe immunostaining method using a marker specific to a differentiatedcell as a target, a Western blot analysis, the RT-PCR method or thelike.

The cell is a cell for prolonging its survival time or a cell forpromoting its proliferation or differentiation by the contact with ECP,and specifically, a nerve cell, bone cell, myocardial cell, fibroblastand the like are preferred. In addition, such a cell may be a primaryculture cell isolated from an animal tissue, passage culture cell,established cell line, or transformed cell into which a foreign gene hasbeen introduced. Such a cell can be examined under the same condition asin a usual animal cell culture. In the case of examining the survival ofa cell, it is preferred that the cell is cultured under a conditionwithout serum or a growth factor. Further, such a cell may be a cellpresent in an individual animal.

The “candidate substance” is, for example, an unknown or known organicor inorganic compound, protein, peptide, polynucleotide, oligonucleotideor the like. In the case where such a candidate substance is broughtinto contact with a cell, a method of adding the candidate substance toa medium of cultured cells, a method of introducing the candidatesubstance into a cultured cell or a cell present in an individual animal(microinjection or an intracellular introduction method using a lipid)or the like can be adopted. In addition, in the case where the candidatesubstance is a polynucleotide or an oligonucleotide, the expressionvector thereof may be transfected into a cell by a known method or anindividual animal may be infected with a virus vector in accordance witha method of gene therapy.

Novel factors specified by this screening method can become an effectivecomponent of a therapeutic composition or a medium composition solely orin combination with ECP.

Hereunder, by showing the results of experiment and research performedin order to confirm the novel physiological activity of ECP as Examples,the invention of this application will be explained in more detail andspecifically, however, the invention of this application is by no meanslimited to the following examples.

EXAMPLES Example 1 Effects of ECP on Proliferation of Cell Line Derivedfrom Normal Tissue

The effects of ECP on the proliferation of a mouse fibroblast cell line(BALB/c 3T3), aortic smooth muscle cell line (A10), mouse mammaryepithelial cell line (HC-11) and human umbilical vessel endothelial cellline (HUVEC) were analyzed in the presence of 10% FBS. First, in DMEMculture medium containing 10% FBS in a 96-well culture plate, each cellline was inoculated at a concentration of 500 cells/well. Then, thecells were cultured for 24 hours in an incubator in which CO₂ wasmaintained at 5%. Then, the respective concentrations of ECP and RNaseA(0 to 10 μM) were added, and the culture was continued for 48 hours inthe same incubator. Then,3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) wasadded, and the proliferation rate of each cell line was obtained.

The results were shown in FIG. 1. As is clear from the FIG. 1, it wasconfirmed that, with regard to BALB/c 3T3 cell line, the cellproliferation was promoted by adding ECP. On the other hand, with regardto the other cell lines, the proliferation was not promoted or theproliferation was inhibited by ECP.

Example 2 Effects of ECP on Proliferation of BALB/c 3T3 Cell

In DMEM culture medium containing 10% FBS in a 96-well culture plate,BALB/c 3T3 A31-K cells were inoculated at a concentration of 1000cells/well, and after 24 hours, ECP or RNaseA was added to the culturesolution to a final concentration of 1 μM, respectively. After theculture was continued for 48 hours, the number of living cells wascounted.

The results are as shown in FIG. 2. ECP significantly promoted the cellproliferation at any concentration. On the other hand, effects of RNaseAon promoting cell proliferation could not be observed.

Example 3 Time-Course Effects of ECP on Proliferation of BALB/c 3T3 Cell

In DMEM culture medium containing 10% FBS in a 96-well culture plate,BALB/c 3T3 cells were inoculated at a concentration of 1000 cells/well.After the cells were cultured for 24 hours, 1 μM ECP or 1 μM RNaseA wasadded to the culture solution to a final concentration of 1 ng/ml,respectively. The culture was continued for 4 days, and the number ofliving cells was counted for every 24 hours. Incidentally, the culturemedium was changed on day 2 of the culture.

The results are as shown in FIG. 3. With regard to the condition of ECPaddition, after the medium was changed on day 2 of the culture, theproliferation of cells was significantly promoted.

Example 4 Effects of ECP on Proliferation of BALB/c 3T3 Cell UnderCondition of Low-Serum Medium

In DMEM culture medium containing 10% FBS in a 24-well culture plate,BALB/c 3T3 cells were inoculated at a concentration of 2×10⁴ cells/well,and cultured. After 24 hours, when the cultured cells became stable, themedium was changed to DMEM culture medium containing 0.5% FBS, then theculture was continued. After 24 hours, 1 μM ECP or RNaseA, and 60 pMbFGF were added to the culture solution in combination, respectively,then the culture was continued for an additional 48 hours. Then, thecells cultured under the respective conditions were observed with aphase-contrast microscope (×20 objective lens).

The results are as shown in FIG. 4. The cells were proliferated withbFGF solely, however, a more significant effect on cell proliferationwas observed under the condition of bFGF+ECP.

Example 5 Effects of ECP on BALB/c 3T3 Cytoskeletal Molecule UnderCondition of Low-Serum Medium

A round cover glass with a diameter of 15 mm which had been sterilizedin advance was placed in a 24-well culture plate, 1× phosphate buffer(PBS) containing 0.1% gelatin was added to each well so that the coverglass was sufficiently dipped in the solution. Then, the plate was leftat room temperature for 30 minutes, whereby the cover glass was coatedwith gelatin. Then, each well was filled with DMEM culture mediumcontaining 10% FBS, and BALB/c 3T3 cells were inoculated at aconcentration of 2×10⁴ cells/well and cultured. After 24 hours, themedium was changed to DMEM culture medium containing 0.5% FBS. After anadditional 24 hours, 1 μM ECP or RNaseA was added to the culturesolution, respectively. After 6 hours, the cover glass on which BALB/c3T3 cells were grown, was taken out from the well, washed 2 to 3 timeswith 1× PBS, then dipped in 4% formaldehyde (Wako Pure ChemicalIndustries, Ltd.) at room temperature for 10 minutes, whereby the cellswere fixed. Then, the cover glass was washed 2 to 3 times with 1× PBS,dipped in 1× PBS containing 0.1% Trton-X100 for 10 minutes, whereby thecell membranes were lysed. Then, the cover glass was washed 2 to 3 timeswith 1× PBS, dipped in 0.5% BSA/PBS and left at room temperature for 30minutes, whereby a nonspecific adsorption region was blocked with BSA inthis operation. A primary antibody was diluted to 11 μg/ml with PBS, andthe cells were dipped in this antibody dilution for 30 minutes toperform a primary antibody reaction. As the primary antibody, threetypes: F-actin, vinculin and a mixture of F-actin and vinculin wereused.

Then, every 5 minutes, the medium was changed and washing was performedwith fresh 1× PBS. As a secondary antibody, RITC-conjugated goatanti-mouse IgG antibody (CHEMICON) and 10 μM FITC-phalloidin/methanol(Molecular Probes) were mixed in 1× PBS so as to yield a finalconcentration of 10 μg/ml and 100 nM, respectively, and the cells weredipped in this antibody dilution for 30 minutes to react with eachother. Then, every 5 minutes, the medium was changed and washing wasperformed with fresh 1× PBS. Then, a solution of an equivalent amount of1× PBS and glycerol was dropped on a slide glass and the foregoing roundcover glass was placed thereon, and observation was performed with aconfocal laser scanning microscope (MRC-1024, Bio-Rad Co).

The results are as shown in FIG. 5. With regard to the cell group in thecase of adding ECP, increase in the formation of many cytoskeletalmolecules (stress fibers) was confirmed (indicated with an arrow).

In addition, by adding 60 pM bFGF as well as 1 μM ECP or RNaseA,observation of cytoskeleton formation was carried out in the samemanner.

The results are as shown in FIG. 6. It was confirmed that cytoskeletalmolecules were more significantly formed in the presence of ECP andbFGF.

Example 6 Effects of ROCK Inhibitor on Cytoskeleton Formation by ECP

BALB/3T3 cells and A31-11-1 cells which is a clone of BALB/3T3 wereinoculated at a concentration of 1×10⁴ cells/well, and cultured in a24-well culture plate. After 24 hours, the medium was changed to DMEMculture medium containing 0.5% FBS, and the culture was continued. Afteran additional 24 hours, 10 μM ROCK inhibitor (Y27632) was added as wellas 1 μM ECP or RNaseA. After 6 hours, the cells were recovered andimmunostained, and cytoskeleton formation was observed with a microscope(×40 objective lens).

The results are as shown in FIG. 7. The promotion of stress fiberformation by ECP was inhibited by adding the ROCK inhibitor, therefore,it was confirmed that Rho-kinase was involved in cytoskeleton formationby ECP.

Example 7 Effects of ECP on Myocardial Cell Derived from Newborn Rat

The cultured myocardial cells were cultured in SFM (2 μM BrDU, 100units/ml penicillin, 100 μg/ml streptomycin) for 48 hours, whereby thecells were dedifferentiated. Then, the culture medium was changed, andECP was added to a final concentration of 100 ng/ml. After an additional24 hours, the cells were washed three times with 1× PBS for 10 minuteseach, then dipped in 4% paraformaldehyde at room temperature for 30minutes, whereby the cells were fixed. Then, the cells were dipped inPBS containing 0.25% Triton-X100 at room temperature for 15 minutes, andreacted in a blocking buffer containing BSA, to which anti-ENH1 antibodyhad been added at a ratio of 1:40, or anti-α-actinin antibody had beenadded at a ratio of 1:100, at 4° C. overnight. Then, a 20-minute washingwith PBS containing 0.03% Triton-X100 was performed and the washing wasrepeated three times. To the blocking buffer, Cy3-labeled anti-mouseantibody and Cy2-labeled anti-rabbit IgG antibody were added (1:250dilution in blocking buffer), respectively, and reaction was performedat 4° C. for 4 hours. After the reaction, a 20-minute washing with PBScontaining 0.03% Triton-X100 was performed again and the washing wasrepeated three times. Then, water content was removed and 90% glycerolwas added.

FIG. 8 shows the results of immunofluorescence staining. As shown inFIG. 8, an effect of ECP on cardiac hypertrophy in a myocardial cell wasconfirmed.

Example 8 Effects of ECP on the Number of Heartbeats of Myocardial CellDerived from Newborn Rat

Myocardial cells isolated from a newborn rat were dedifferentiated inthe same manner as in Example 7 and ECP was added to the culturesolution (10 ng, 100 ng or 1 μg per ml). Twenty-four hours after theaddition, the number of heartbeats was counted.

The results are as shown in FIG. 9. It was confirmed that the number ofheartbeats of myocardial cell was increased in a dose-dependent mannerby ECP.

Further, the effects of a ROCK inhibitor (Y27632) on the effects of ECPon increase in the number of heartbeats of myocardial cell were studied.The adding amounts of Y27632 were set at 0, 5, 10 and 15 μM, and the ECPconcentration was set at 100 ng/ml.

The results are as shown in FIG. 10. The increase in the number ofheartbeats due to ECP was not observed by adding the ROCK inhibitor,therefore, it was confirmed that this ECP activity depended on theRho-kinase pathway in the signal transduction cascade.

Example 9 Effects of ECP on the Survival of Nerve Like PC12 Cell UnderCondition of Serum-Free Medium

PC12 cells were cultured overnight in a 96-well culture plate at 1×10⁴cells/well/100 μl. As the culture medium, DMEM culture medium containing10% FBS, penicillin, and streptomycin was used. Then, washing wasperformed once with 150 μl of DMEM, and the medium was changed to DMEMculture medium containing 100 μl of ECP, penicillin, and streptomycin.After cells were cultured for 72 hours, the number of living cells wascounted using Cell Titer-GloTM Luminescent Cell Viability Assay (No.G7570; Promega).

The results are as shown FIG. 11. It was confirmed that by adding ECP,the survival of nerve cells was maintained.

Example 10 Effects of ECP on Alkali Phosphatase (ALP) Activity inOsteoblast-Like MC3T3-E1 Cell

MC3T3-E1 cells were inoculated into α-MEM medium in a 24-well cultureplate, and ECP (at a final concentration of 1 ng, 10 ng, 100 ng or 1 μgper ml), BMP-4 (100 ng/ml) or bFGF (100 ng/ml) was added. After 72hours, the cells were washed with 10 mM Tris-HCl (pH 7.2) solutiontwice, and lysed in 10 mM Tris-HCl (pH 7.2) solution containing 0.1%Triton-X100. Then, the cell lysis solution was subjected to sonicationtreatment for 15 seconds to homoginize the cells. Then, 100 μl of thehomogenized cell solution and 0.1 M aminomethyl propanol were mixed with100 μl of a substrate solution (4 mg/ml p-nitorophenyl phosphate and 2mM MgCl₂) adjusted at pH 10.5, and they were reacted with each other at37° C. for 30 minutes. The reaction was terminated with 0.5 M NaOH, andthe absorbance at 410 nm was measured. The obtained value was divided bythe total amount of proteins in the homogenized cell solution, wherebythe specific activity was calculated.

The results are as shown in FIG. 12. The value of the control wasdefined as 100%, and the effects of the respective added components areshown. In the case of adding ECP at 1 ng/ml, the ALP activity equal toor higher than that of BMP-4 which is a bone-forming factor wasobtained.

INDUSTRIAL APPLICABILITY

As described in detail above, according to the invention of thisapplication, a therapeutic composition based on a novel physiologicalactivity of ECP is provided, and a new way to cure a heart disease, bonedisease, neurodegenerative disease or the like caused by an aberrationin the survival, proliferation and/or differentiation of a cell will bedeveloped. In addition, a medium composition for prolonging the survivaltime, promoting proliferation and/or promoting differentiation of acultured cell is provided. Further, a method for specifying a novelfactor for promoting the survival, proliferation and/or differentiationof a cell is provided, whereby it becomes possible to develop a moreeffective therapeutic composition.

1. A composition which is a therapeutic composition for a disease causedby a failure in the survival, proliferation and/or differentiation of acell characterized by containing eosinophil cationic protein andpharmacological components.
 2. The composition according to claim 1,wherein the disease caused by a failure in the survival, proliferationand/or differentiation of a cell is a heart disease, bone disease orneurodegenerative disease.
 3. A composition which is a mediumcomposition for promoting the survival, proliferation and/ordifferentiation of a cell containing eosinophil cationic protein and acell biological component.
 4. A screening method, which is a method ofscreening an active ingredient substance of a therapeutic compositionfor a disease caused by a failure in the survival, proliferation and/ordifferentiation of a cell, characterized by bringing a candidatesubstance into contact with a cell and specifying, as a targetsubstance, a substance for promoting the survival and/or differentiationof a cell at the same level or higher than eosinophil cationic protein.5. The screening method according to claim 4, wherein the cell is anerve cell, bone cell, myocardial cell or fibroblast.